Apoptosis in normal lymphoid organs from healthy normal, conventional pigs at different ages detected by TUNEL and cleaved caspase-3 immunohistochemistry in paraffin-embedded tissues

The frequency and the distribution of apoptotic cells were investigated in formalin-fixed paraffin-embedded lymphoid tissues from healthy conventional pigs at four different ages (6 days, 2 months, 3.5 months and 5 months). Samples of tonsil, mesenteric lymph node, spleen, thymus and Peyer's patches were histologically processed and apoptosis evaluated with the TUNEL reaction and cleaved caspase-3 immunohistochemistry. In each technique, quantification of positive labelling was done for each particular lymphoid tissue area. The labelling pattern and distribution were similar for TUNEL and cleaved caspase-3. TUNEL stained mainly apoptotic bodies inside macrophages, but signal was also seen in free apoptotic bodies and in the nuclei of lymphocyte-like cells. The anti-cleaved caspase-3 antibody labelled mainly nuclei of lymphocyte-like cells. All tissues presented a similar distribution pattern of apoptosis, except for the 6-day-old group. In this group, a scattered distribution of positive cells was detected in tonsil, lymph node and spleen. In the tonsil and mesenteric lymph nodes from the older pigs, follicular areas presented higher amounts of positive cells than interfollicular areas. Moreover, the splenic white pulp showed more positive reaction than the red pulp, especially when they included germinal centres. In all groups, the follicular areas of ileal Peyer's patches presented more labelled cells than the dome and the lamina propria. In the thymus, the higher apoptotic rates were found in the cortex. In general, TUNEL yielded higher rates of positive cells than cleaved caspase-3 immunolabelling. A good correlation between the two techniques was found for thymus, tonsil and mesenteric lymph node, but not for Peyer's patches and spleen. This study describes a detailed histochemical characterization of apoptosis in pig lymphoid tissues using TUNEL and a cleaved caspase-3 immunolabelling at different ages. Moreover, our results indicate that TUNEL and cleaved caspase-3 techniques can be equivalent only when tissues have a high or low levels of apoptosis, since a considerable discrepancy was found in intermediate situations. Data from this study should be useful for future comparative studies under disease conditions.     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

Apoptosis induced in vivo by new type gosling viral enteritis virus

In this study, apoptosis was induced by new type gosling viral enteritis virus (NGVEV) in experimentally infected goslings is reported in detail for the first time. After 3-day-old goslings were orally inoculated with a NGVEV-CN strain suspension, the time course of NGVEV effects on apoptotic morphological changes of the internal tissues was evaluated. These changes were observed by histological analysis with light microscopy and ultrastructural analysis with transmission electron microscopy. DNA fragmentation was assessed with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and DNA ladder analysis. A series of characteristic apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, plasma membrane blebbing, and formation of apoptotic bodies were noted. Apoptosis was readily observed in the lymphoid and gastrointestinal organs, and sporadically occurred in other organs after 3 days post-infection (PI). The presence and quantity of TUNEL-positive cells increased with infection time until 9 days PI. DNA extracted from the NGVEV-infected gosling cells displayed characteristic 180~200 bp ladders. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages, monocytes, and epithelial and intestinal cells. Necrosis was subsequently detected during the late NGVEV-infection phase, which was characterized by cell swelling, plasma membrane collapse, and rapidly lysis. Our results suggested that apoptosis may play an important role in the pathogenesis of NGVE disease.     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Tumor necrosis factor-alpha induces apoptosis in cultured porcine luteal cells

Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.     

Suppressive effect of hyaluronan on chondrocyte apoptosis in experimentally induced acute osteoarthritis in dogs

Dogs receiving anterior cruciate ligament transection (ACLT) were treated with either intravenous (IV) or intraarticular (IA) administration of hyaluronan (HA), and differences in appearance of chondrocyte apoptosis of the stifle joint were investigated. Chondrocyte apoptosis was detected using flow cytometry as well as by staining with TdT-mediated dUTP nick end labeling (TUNEL). The percentage of apoptotic chondrocytes in dogs with ACLT was significantly higher than that in intact (non-ACLT) dogs. Dogs treated with IA or IV injection of HA after ACLT had fewer apoptotic chondrocytes than non-treated dogs after ACLT. It was suggested that ACLT-induced apoptosis of chondrocytes was suppressed by HA administration of either IA or IV.     

Pathogenesis of type 1 (European genotype) porcine reproductive and respiratory syndrome virus in male gonads of infected boar

The objective of this study was to determine the pathogenesis of experimental infection with a type 1 porcine reproductive and respiratory syndrome virus (PRRSV) by defining the sites of viral replication and apoptosis in male gonads from infected boars for a period of 21 days after intranasal inoculation. Microscopically, hypospermatogenesis and abundant germ cell depletion and death were observed in the testes. Such germ cell death occurs by apoptosis, as determined by a characteristic histological patterns and evidence of massive DNA fragment detected in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) reaction. PRRSV was detected in the testicular tissue of infected boars only. Viral nucleic acid was localized in spermatogonia, spermatocytes and spermatids but not in the vesicular and bulbourethral gland. In serial sections, PRRSV-positive cells did not co-localized with apoptotic cells. TUNEL-positive apoptotic cells were more numerous than PRRSV-positive cells in testicular sections. The present study demonstrated that type 1 PRRSV infects the spermatogonia and their progeny, and induces apoptosis in these germ cells.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Lymphoid apoptosis in acute canine distemper

The relationship between the canine distemper virus (CDV) infection and apoptosis in the canine lymphoid tissues was investigated using immunostaining for single stranded DNA (ssDNA), TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method, and electron microscopy. Twenty-six lymphoid tissues from 8 spontaneously CDV-infected dogs and 1 non-infected dog were used, and lesions were classified into 4 groups according to frequency of the CDV-antigen. Histologically, the degree of lymphoid depletion tended to depend on amount of CDV antigen. The numbers of ssDNA- and TUNEL-labeling cells were significantly high in the lymphoid tissues with abundant viral antigen. However, ssDNA- and TUNEL-positive lymphocytes were also frequently found even in the lymphoid tissues where there was only a small amount of CDV-antigen in sinus histiocytes. The incidence and distribution of apoptotic cells in the CDV-antigens-negative lymphoid tissues from infected dogs were equal to those from a non-infected dog. Double labeling immunostaining using a ssDNA and a CDV nucleocapsid protein (CDV-NP) antibody revealed that there were ssDNA positive but CDV-NP negative cells besides those stained doubly positive. Ultrastructurally, lymphocytes in the CDV-infected lymphoid tissues frequently had characteristic morphological features of apoptosis such as apoptotic bodies. All these results suggest that CDV leads to lymphocytic apoptosis directly or indirectly, resulting in severe lymphoid depletion and immunosuppression in acute or subacute phase of CDV infection.     

Apoptosis in feline panleukopenia and canine parvovirus enteritis

Tissue samples of cats and dogs with panleukopenia and parvovirus enteritis, respectively, were examined for the presence of viral antigen-positive cells and apoptotic cells by immunohistochemistry and by TUNEL assay (Terminal Transferase-Mediated dUTP Nick End Labelling). Compared to control animals, infected cats and dogs generally had more TUNEL-positive cells. Cell types positive for parvovirus antigen, for example digestive tract epithelial and mesenchymal cells, and lymphocytes and macrophages in lymphoid tissues were also positive for TUNEL signals. Occasionally, TUNEL signal and viral antigen were present in the same tissue areas, suggesting a direct viral trigger of apoptosis. More frequently, however, there was no complete overlap of antigen and TUNEL-positive areas. The results of this study indicate that apoptotic cell death contributes significantly to the widespread tissue damage of parvovirus infection in cats and dogs.     

Developmental changes in cell proliferation and apoptosis in the normal duck thymus

Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.     

Hepatocyte apoptosis in dairy cattle during the transition period

The objective of this study was to investigate hepatocyte apoptosis in dairy cows during the transition period. Four clinically healthy, pregnant dairy cattle were used. The cows had no clinical diseases throughout this study. Blood samples were collected and livers were biopsied from the cows at 3 different times: 3 weeks before expected partition (wk −3); during parturition (wk 0), and 3 weeks (wk +3) after parturition. The damage to deoxyribonucleic acid (DNA) caused by hepatocytes was evaluated by comet assay. The apoptotic features of hepatocytes were examined by immunohistochemistry and electron microscopic analyses. The hepatic triglyceride content markedly increased at wk 0 and wk +3 compared with the values at wk −3. The results of the comet assay showed increases in the mean tail moment values of hepatic cells after parturition in all cows, which suggested increased DNA damage. Histopathologically, the hepatocytes began to contain lipid droplets at wk 0 and were severely opacified at wk +3. Caspase-3-positive and single-stranded DNA-(ssDNA)-positive cells were first detected in the liver after parturition. Condensation of nuclear chromatin, a typical sign of apoptosis, was confirmed by transmission electron microscopy after parturition. These results suggest that apoptosis is induced in hepatocytes of dairy cows around parturition and may result from lipotoxicity in hepatocytes.     

Distribution of apoptotic cells and apoptosis-related molecules in the developing murine palatine rugae

Distribution of apoptotic cells and expression of the apoptosis-related factors p53, bcl-2 and bad during morphogenesis of the murine palatine rugae (PR) were examined histochemically using the terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) technique and specific antibodies against apoptosis and cell cycle-related molecules. Formation of the PR rudiment was controlled by cell proliferation and apoptosis in the palatal epithelium. TUNEL-positive cells were detected only at the epithelial placode area at 12.5-13.5 days post coitus (dpc), but only a few cells were positive at the protruding PR area at 14.5-16.5 dpc. Bcl-2 protein was expressed mainly in the areas outside of those containing TUNEL-positive cells at 15.5 -6.5 dpc. P53 protein was not detected throughout gestation. Bad was detected in the epithelial layer at 13.5 and 15.5 dpc and overlapping the apoptotic area at 13.5-15.5 dpc. Apoptosis of palatal epithelial cells might therefore involve spatiotemporally regulated expression of bad during murine PR development.     

Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs

Porcine circovirus type 2 (PCV2) is clinically associated with respiratory disease, failure-to-thrive, hepatitis, and diarrhea; however, the precise pathogenesis of PCV2-associated disease and in particular its involvement in apoptosis is still controversial. The objectives of this study were (1) to determine whether PCV2 is associated with apoptosis by examining and comparing hepatic tissues from clinically affected or unaffected gnotobiotic pigs that were experimentally infected with PCV2, (2) to determine if there are differences between PCV2a and PCV2b in inducing hepatocyte apoptosis, and (3) to determine if there are differences between apoptosis detection systems. Forty-eight gnotobiotic pigs were separated into five groups based on inoculation status and development of clinical disease: (1) sham-inoculated, clinically-unaffected (n=4), (2) inoculated with PCV2a, clinically-unaffected (n=10), (3) inoculated with PCV2a, clinically-affected (n=6), (4) inoculated with PCV2b, clinically-unaffected, (n=13) and (5) inoculated with PCV2b, clinically-affected (n=15). Formalin-fixed, paraffin-embedded sections of liver from all pigs were analyzed for signs of apoptosis [presence of single strand DNA breaks in the nucleus by the terminal transferase dUTP nick end labeling (TUNEL) assay or presence of intra-nuclear cleaved caspase 3 (CCasp3) demonstrated by CCasp3 immunohistochemistry (IHC)]. In addition, the liver tissues were also tested for presence of cytoplasmic and intra-nuclear PCV2 antigen by an IHC assay. Specific CCasp3 and TUNEL labeling was detected in the nucleus of hepatocytes in PCV2a and PCV2b infected pigs with significantly (P<0.05) higher levels of apoptotic cells in clinically-affected pigs. Regardless of PCV2 subtype (PCV2a; PCV2b), there were higher levels of PCV2 antigen in clinically-affected pigs compared to clinically-unaffected pigs. There was no significant difference in detection rate of apoptotic cells between the TUNEL assay and CCasp3 IHC. When high amounts of PCV2 antigen were present, the incidence of CCasp3 and TUNEL staining also increased regardless of the PCV2 genotype. This suggests that PCV2-induced apoptosis of hepatocytes is important in the pathogenesis of PCV2-associated lesions and disease.     

Temporary inhibition of neuronal apoptosis in Aujeszky's disease virus-infected swine

Previous studies have shown that during acute infection of the porcine trigeminal ganglia (TG), Aujeszky's disease virus (ADV)-infected neurons are protected from apoptosis induced by the virus itself and by cells of the immune system. However, TG neurons productively infected by ADV finally die and are phagocytosed by adjacent cells, a fact that leads us to speculate that the inhibition of neuronal apoptosis by ADV may be temporary rather than absolute. To address this issue we used TG and brain stem from pigs during acute infection by ADV. Infected cells were detected by immunohistochemical staining of viral antigens, whereas apoptotic cells were identified with an anti-active caspase-3 antibody, the TUNEL assay and by transmission electron microscopy. The results obtained in this study support the contention that the inhibition of neuronal apoptosis by ADV is temporary, since activation of caspase-3 could be detected in infected neurons at late stages in infection and because foci of advanced neuronophagia contained neurons exhibiting typical ultrastructural features of apoptosis.     

水牛胎儿卵泡细胞和卵母细胞凋亡的特征

选择5月龄胎儿水牛的卵巢，采用石蜡切片、HE染色的光镜观察和DNA末端原位标记技术（TUNEL）进行了研究。结果表明：5月龄胎儿卵巢上分布着大量的凋亡细胞；在皮质部存在的大量腔前卵泡中，均匀分布有单个凋亡的卵泡细胞和卵母细胞，凋亡细胞的主要组织学变化是细胞缩小，核浓缩；DNA末端标记变化主要显示出TUNEL阳性。上述结果提示：水牛卵泡细胞和卵母细胞凋亡在胎儿时期已经存在，细胞凋亡是启动水牛腔前卵泡闭锁的潜在机理。     

Apoptotic Cell Localization in Preantral and Antral Follicles in Relation to Non‐cyclic and Cyclic Gilts

The objective of this study was to determine apoptotic cell localization in preantral and antral follicles of porcine ovaries. Additionally, the proportion of cells undergoing apoptosis was also compared between delayed puberty gilts and normal cyclic gilts. Ovarian tissues were obtained from 34 culled gilts with age and weight of 270.1 ± 3.9 days and 143.8 ± 2.4 kg, respectively. The gilts were classified according to their ovarian appearance as ‘non‐cyclic’ (n = 7) and ‘cyclic’ (n = 27) gilts. The terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assay was used to determine apoptotic cell expression in different compartments of the ovarian tissue sections. All apparent preantral (n = 110) and antral (n = 262) follicles were evaluated using image analysis software. It was found that apoptotic cells were expressed in both granulosa (22.2%) and theca cell layers (21.3%) of the follicles in the porcine ovaries. The proportion of apoptotic cells in the granulosa layer in the follicles was positively correlated with that in the theca layer (r = 0.90, p < 0.001). Apoptosis did not differ significantly between preantral and antral follicles in either granulosa (27.8% and 26.4%, p > 0.05) or theca cell layers (28.6% and 26.5%, p > 0.05). The proportion of apoptotic cells in non‐cyclic gilts was higher than cyclic gilts in both granulosa (31.7% and 22.6%, p < 0.001) and theca cell layers (34.8% and 20.2%, p < 0.001). This study indicated that apoptosis of the granulosa and theca cell layers in the follicles was more pronounced in the ovarian tissue of delayed puberty gilts than cyclic gilts. This implied that apoptosis could be used as a biologic marker for follicular development/function and also that apoptosis was significantly associated with anoestrus or delayed puberty in gilts, commonly observed in tropical climates.     

阿维菌素对体外培养王鸽脑神经细胞凋亡和一氧化氮水平的影响

本试验以阿维菌素染毒原代培养的王鸽脑神经细胞为模型,观察阿维菌素对神经细胞凋亡的毒性作用和一氧化氮产生量的影响。采用原位末端标记法观察细胞凋亡,四唑盐比色法检测神经细胞相对存活率,流式细胞术检测凋亡细胞百分比,分光光度法测定细胞一氧化氮的产生量。结果显示,阿维菌素可诱导神经细胞凋亡和细胞坏死,并具有随着阿维菌素剂量递增而增加的趋势,同时阿维菌素也能引起一氧化氮产生量降低,但是未发现存在剂量效应关系。     

The replication of canine herpesvirus (CHV) induces apoptosis in canine kidney cell line: short communication

The alphaherpesvirus canine herpesvirus (CHV) was tested in order to determine whether or not it has apoptotic potential. We have demonstrated that lytic replication of CHV resulted in induction of apoptosis. This phenomenon was confirmed using different techniques including in situ TUNEL assay and DNA laddering. The apoptotic activity of CHV might influence the pathobiology of this virus.